1. Field of the Invention
The invention is directed to a beam deflecting unit for multiple-axis examination of specimens in a microscope which, on the one hand, enables the illumination of the specimen by one or more beams from several sides and/or observation of the specimen by one or more beams from several sides and, on the other hand, allows observation of the specimen at an angle to the illumination axis. The beam deflecting unit according to the invention can be used in available far-field raster scanning light microscopes and is situated between the microscope objective and the focal plane. It is adapted to the size ratios of the specimen and of the free working length of the objective.
2. Description of the Related Art
Technical scientific literature discloses confocal scanning light microscopes which have a resolution along the optical axis (axial resolution) and can generate images with an appreciably reduced depth of focus. A problem arises. in that the microscope objectives detect only a part of the total solid angle and accordingly detect only a portion of the light which is radiated in all directions from a point. As a result, the axial resolution is at least three-times worse than the axial resolution. Generally, the ratio is greater.
For an additionally increased resolution in the axial direction, a double-confocal scanning light microscope that was suggested in DE-OS 40 40 441 is characterized by the use of a second objective on the other side of the object plane, wherein both objectives illuminate a common object point simultaneously and/or detect the light proceeding from it. In the case of a coherent illumination of the object by the two objectives, the observation volume is reduced by interference along the optical axis. The reduction in observation volume is synonymous with an improvement in resolution.
Further, DE-OS 43 26 4731 discloses a scanning light microscope in which at least two objectives are arranged in such a way that they illuminate an object point simultaneously and/or collect the light proceeding from the object point, wherein at least two of the objectives do not lie on a common axis. In addition, as is the case in a double-confocal scanning light microscope, means for changing interference can be arranged in such a way that light passing through one of the objectives is coherently superposed at the object and/or on at least one of the light detectors with light passing through one of the other objectives at the object so that it interferes.
In the scanning light microscope known from DE-OS 43 26 473, two objectives are preferably arranged in such a way that their axes are perpendicular to one another, and one objective is used for the illumination of the specimen, while the second objective is used for the observation of the specimen. An improved resolution is achieved due to the fact that the illumination axis which is determined by the optical axis of the objective used for illumination is arranged perpendicular to the observation axis which is determined by the optical axis of the objective used for observation.
The high resolution is brought about in the following manner: The intensity distribution in the focal range is described by the illumination point spread function (PSF). The detection probability for the light proceeding from the focal range is described by the observation PSF when using a detection pinhole diaphragm. The PSF of a confocal microscope is the product of the illumination PSF and the observation PSF. The more extensive or spread out it is, the poorer the resolution of the microscope. Due to the approximately perpendicular arrangement of the illumination axis and the observation axis relative to one another, the large extension of the illumination PSF along its axis is compensated for by the small extension of the illumination PSF along this axis, so that a resolution of approximately equal quality is achieved along all three spatial axes.
According to another embodiment form of the scanning light microscope known from DE-OS 43 26 473, three objectives are arranged in such a way that the axes of two objectives are perpendicular to one another, while the axis of the third objective lies on the axis of one of the other two objectives. In this case, the light passing through one of the objectives is coherently superposed on the object and/or on one of the light detectors with light passing through one of the other objectives, so that it interferes. In the case of interference of the illumination light, the high resolution is brought about in the following manner: The illumination PSF is modulated by an interference pattern due to interference along the illumination axis of the two illumination beams entering from opposite directions, insofar as the illumination beams are coherent. If the phase difference between the two illumination partial beams in the focal point is equal to zero or is a whole-number multiple of 21, the interference in the geometric focal point is structural and the illumination PSF is formed of a spatially sharply delimited principal maximum and a plurality of secondary maxima along the illumination axis which would reduce resolution if they could not be suppressed. The confocal PSF which is the product of the illumination PSF and the observation PSF is also modulated by the interference along the illumination axis. However, the secondary maxima are suppressed by the vertical observation through the third objective, so that the confocal PSF has an axial extension which is essentially determined by that of the principal maximum of the illumination PSF. This is synonymous with a high resolution.
Further, a double-objective system with two objectives is known from the German Patent Application 196 29 725.7, wherein one objective serves to focus illumination light and the other serves to collect observation light. The observation axis of the second objective is at an angle to the illumination axis of the first objective. In this way, according to the optical principle described in DE-OS 43 26 473, an improved resolution is achieved. In contrast to the construction described in DE-OS 43 26 473 with complete separation of the illumination beam paths and observation beam paths, the double-objective system from Patent Application 196 29 725.7 enables the realization of the optical principle described in DE-OS 43 26 473 in an available light microscope. In this case, the customary individual objective in the conventional light microscope is exchanged for the double-objective system.
In contrast, the present invention shows a way in which the optical principle described in DE-OS 43 26 473 can be realized in an available scanning light microscope with only one objective.